Methods to detect and analyze early apoptotic cell death in brain are important in developing and testing efficient therapeutic interventions. The long-term objective of this proposal is to develop a new approach for visualization of early apoptosis in tissue sections and to use it to assess the dynamics of apoptosis initiation during brain ischemia. Specific Aims of this project are: 1) To develop a new technique for detection of initial stages of apoptosis in situ. 2) Use the newly developed approach to analyze the dynamics of early apoptosis in rat brain after experimental stroke. The approach will detect a specific fingerprint of topoisomerase II, whose involvement in early apoptosis was recently demonstrated. The method will use the unique enzymatic properties of vaccinia topoisomerase to label characteristic DNA breaks produced by topoisomerase II during the initial stages of apoptosis. The previously undetectable type of double-strand DNA breaks with 4 base-long protruding 5'OH ends, produced by cellular topoisomerase II at the start of apoptosis, will be selectively visualized. The technology will be tested on different models of apoptotic and nonapoptotic DNA damage, and will then be applied to study focal brain ischemia in rats. This new assay will have a single cell sensitivity limit and will be an important addition to the apoptosis detection arsenal, filing the gap in techniques for labeling early apoptosis in the tissue section format. It will permit better evaluation of ischemic cellular damage and will be helpful in development of new therapies for ischemic brain damage. It will also have a broad applicability in all fields of biomedicine where detection of early apoptosis is important. [unreadable] [unreadable] [unreadable]